Efficacy of Supra-HFR in Removing FGF23 and Cytokines: A Single Session Analysis

Background/Aim: Supra hemodiafiltration with reinfusion of the endogenous ultrafiltrate (Supra-HFR) is a dialysis technique used to improve uremic toxin removal in the range of the middle molecular weight molecules. Supra-HFR does not require the preparation and online infusion of high purity dialysis water because it allows the production of an endogenous ultrafiltrate that undergoes detoxification through an adsorbing resin. Patients and Methods: We investigated the ability of Supra-HFR to remove fibroblast growth factor 23 (FGF23), interleukin 6 (IL-6), tumor necrosis factor alpha (TNFalpha), interleukin 8 (IL-8), and transforming growth factor alpha (TGF-alpha) after a single session dialysis in nine patients affected by end stage renal disease (ESRD). The same patients underwent a single session of online hemodiafiltration (OLHDF) to evaluate possible differences in FGF23 and IL-6 levels. Results: A significant reduction in FGF23 was observed with both Supra-HFR (p=0.001) and OL-HDF. As for TNF-alpha and TGF-alpha, which were measured using Supra-HFR only, their percentage values were significantly lower at the end of dialysi

Endothelial follistatin-like-1 regulates the postnatal development of the pulmonary vasculature by modulating BMP/Smad signaling

Bone morphogenetic protein (BMP) signaling regulates vascular smooth muscle maturation, endothelial cell proliferation, and tube formation. The endogenous BMP antagonist Follistatin-like 1 (Fstl1) is highly expressed in pulmonary vascular endothelium of the developing mouse lung, suggesting a role in pulmonary vascular formation and vascular homeostasis. The aim of this study was to investigate the role of Fstl1 in the pulmonary vascular endothelium. To this aim, Fstl1 was conditionally deleted from endothelial and endothelial-derived cells using Tie2-cre driven Fstl1-KO mice (Fstl1-eKO mice). Endothelial-specific Fstl1 deletion was postnatally lethal, as ∼70% of Fstl1-eKO mice died at three weeks after birth. Deletion of Fstl1 from endothelium resulted in a reduction of right ventricular output at three weeks after birth compared with controls. This was associated with pulmonary vascular remodeling, as the percentage of actin-positive small pulmonary vessels was increased at three weeks in Fstl1-eKO mice compared with controls. Endothelial deletion of Fstl1 resulted in activation of Smad1/5/8 signaling and increased BMP/Smad-regulated gene expression of Jagged1, Endoglin, and Gata2 at one week after birth compared with controls. In addition, potent vasoconstrictor Endothelin-1, the expression of which is driven by Gata2, was increased in expression, both on the mRNA and protein levels, at one week after birth compared with controls. At three weeks, Jagged1 was reduced in the Fstl1-eKO mice whereas Endoglin and Endothelin-1 were unchanged. In conclusion, loss of endothelial Fstl1 in the lung is associated with elevated BMP-regulated genes, impaired small pulmonary vascular remodeling, and decreased right ventricular output

Early Embryonic Vascular Patterning by Matrix-Mediated Paracrine Signalling: A Mathematical Model Study

During embryonic vasculogenesis, endothelial precursor cells of mesodermal origin known as angioblasts assemble into a characteristic network pattern. Although a considerable amount of markers and signals involved in this process have been identified, the mechanisms underlying the coalescence of angioblasts into this reticular pattern remain unclear. Various recent studies hypothesize that autocrine regulation of the chemoattractant vascular endothelial growth factor (VEGF) is responsible for the formation of vascular networks in vitro. However, the autocrine regulation hypothesis does not fit well with reported data on in vivo early vascular development. In this study, we propose a mathematical model based on the alternative assumption that endodermal VEGF signalling activity, having a paracrine effect on adjacent angioblasts, is mediated by its binding to the extracellular matrix (ECM). Detailed morphometric analysis of simulated networks and images obtained from in vivo quail embryos reveals the model mimics the vascular patterns with high accuracy. These results show that paracrine signalling can result in the formation of fine-grained cellular networks when mediated by angioblast-produced ECM. This lends additional support to the theory that patterning during early vascular development in the vertebrate embryo is regulated by paracrine signalling

Cells derived from the coelomic epithelium contribute to multiple gastrointestinal tissues in mouse embryos.

Gut mesodermal tissues originate from the splanchnopleural mesenchyme. However, the embryonic gastrointestinal coelomic epithelium gives rise to mesenchymal cells, whose significance and fate are little known. Our aim was to investigate the contribution of coelomic epithelium-derived cells to the intestinal development. We have used the transgenic mouse model mWt1/IRES/GFP-Cre (Wt1(cre)) crossed with the Rosa26R-EYFP reporter mouse. In the gastrointestinal duct Wt1, the Wilms' tumor suppressor gene, is specific and dynamically expressed in the coelomic epithelium. In the embryos obtained from the crossbreeding, the Wt1-expressing cell lineage produces the yellow fluorescent protein (YFP) allowing for colocalization with differentiation markers through confocal microscopy and flow cytometry. Wt1(cre-YFP) cells were very abundant throughout the intestine during midgestation, declining in neonates. Wt1(cre-YFP) cells were also transiently observed within the mucosa, being apparently released into the intestinal lumen. YFP was detected in cells contributing to intestinal vascularization (endothelium, pericytes and smooth muscle), visceral musculature (circular, longitudinal and submucosal) as well as in Cajal and Cajal-like interstitial cells. Wt1(cre-YFP) mesenchymal cells expressed FGF9, a critical growth factor for intestinal development, as well as PDGFRα, mainly within developing villi. Thus, a cell population derived from the coelomic epithelium incorporates to the gut mesenchyme and contribute to a variety of intestinal tissues, probably playing also a signaling role. Our results support the origin of interstitial cells of Cajal and visceral circular muscle from a common progenitor expressing anoctamin-1 and SMCα-actin. Coelomic-derived cells contribute to the differentiation of at least a part of the interstitial cells of Cajal

Follistatin-like 1 in development and human diseases

Follistatin-like 1 (FSTL1) is a secreted glycoprotein displaying expression changes during development and disease, among which cardiovascular disease, cancer, and arthritis. The cardioprotective role of FSTL1 has been intensively studied over the last years, though its mechanism of action remains elusive. FSTL1 is involved in multiple signaling pathways and biological processes, including vascularization and regulation of the immune response, a feature that complicates its study. Binding to the DIP2A, TLR4 and BMP receptors have been shown, but other molecular partners probably exist. During cancer progression and rheumatoid arthritis, controversial data have been reported with respect to the proliferative, apoptotic, migratory, and inflammatory effects of FSTL1. This controversy might reside in the extensive post-transcriptional regulation of FSTL1. The FSTL1 primary transcript also encodes for a microRNA (miR-198) in primates and multiple microRNA-binding sites are present in the 3′UTR. The switch between expression of the FSTL1 protein and miR-198 is an important regulator of tumour metastasis and wound healing. The glycosylation state of FSTL1 is a determinant of biological activity, in cardiomyocytes the glycosylated form promoting proliferation and the non-glycosylated working anti-apoptotic. Moreover, the glycosylation state shows differences between species and tissues which might underlie the differences observed in in vitro studies. Finally, regulation at the level of protein secretion has been described

Antibodies used in this study.

<p>Antibodies used in this study.</p

Contribution of the Wt1^cre-YFP cells to the visceral smooth muscle and Cajal-like interstitial cells.

<p>A. Colocalization of SMCα-actin with YFP in cells ventral to the endoderm at the stage E11.5 (arrows). SMCα-actin+/Wt1^cre-YFP cells can be seen in the wall of a large vessel (arrowhead). B,C. Colocalization of SMCα-actin with YFP in visceral circular muscle layer (CL) at E15.5, in transverse and longitudinal section, respectively. A submesothelial cell, positive for both markers, is shown in C by the arrowhead. Note the YFP-positive vessel wall (arrow in C). D, E. The intestinal coelomic epithelium is RALDH2 immunoreactive by E13.5 (D) and E15.5 (E). Note the presence of SMCα-actin immunoreactive cells, probably progenitors of the longitudinal muscle layer, lying behind the RALDH2+ mesothelium in E. F. In this neonate, colocalization of YFP with SMCα-actin is observed in the circular muscular layer (arrows). CD34+ cells are abundant in the submucosal layer, showing thin prolongations. Some of these cells are also Wt1^cre-YFP (arrowheads). Note the presence of submucosal SMCα-actin cells forming the innermost muscle layer. CD34 does not colocalize with SMCα-actin. Scale bars = 25 µm.</p

Vascular contribution of the Wt1-expressing cell lineage.

<p>A–C. Colocalization of the endothelial markers Pecam-1 (A, B) and CD34 (C) with YFP in intestinal vessels at the stage E11.5. D. Colocalization of the pericyte marker NG2 with YFP at the stage E13.5. E. Immunolocalization of the fibroblastic marker 5B5 at the stage E18.5. This marker does not colocalize with YFP. F. Immunolocalization of the fibroblastic marker FSP1 in a neonate. Colocalization with YFP is not observed. G. Analysis of dissociated intestines from an E11.5 embryo by flow cytometry. In this representative experiment, colocalization of PECAM-1 with YFP (arrow) was found in a 0.3% of the total cells, and in 2.9% of the Wt1^cre-YFP cells. Scale bars A, E, F = 50 µm; B,C,D = 25 µm.</p

Identifying pathogenic variants in the Follistatin-like 1 gene (FSTL1) in patients with skeletal and atrioventricular valve disorders

Background: Follistatin-like 1 (Fstl1) is a glycoprotein expressed throughout embryonic development. Homozygous loss of Fstl1 in mice results in skeletal and respiratory defects, leading to neonatal death due to a collapse of the trachea. Furthermore, Fstl1 conditional deletion from the endocardial/endothelial lineage results in postnatal death due to heart failure and profound atrioventricular valve defects. Here, we investigated patients with phenotypes similar to the phenotypes observed in the transgenic mice, for variants in FSTL1. Methods: In total, 69 genetically unresolved patients were selected with the following phenotypes: campomelic dysplasia (12), small patella syndrome (2), BILU (1), and congenital heart disease patients (54), of which 16 also had kyphoscoliosis, and 38 had valve abnormalities as their main diagnosis. Using qPCR, none of 69 patients showed copy number variations in FSTL1. The entire gene body, including microRNA-198 and three validated microRNA-binding sites, were analyzed using Sanger sequencing. Results: No variants were found in the coding region. However, 8 intronic variants were identified that differed significantly in their minor allele frequency compared to controls. Variant rs2272515 was found to significantly correlate (p < 0.05) with kyphoscoliosis. Conclusion: We conclude that pathogenic variants in FSTL1 are unlikely to be responsible for skeletal or atrioventricular valve anomalies in humans

Presence of Wt1^cre-YFP cells in the endoderm.

<p>A. Fibronectin immunoreactivity is high in the prospective submucosal area. Note the presence of Wt1^cre-YFP cells inside the endodermal mucosa, as well as into the intestinal lumen. Many luminal cells show pycnotic nuclei suggesting cell death. B-D. Sections obtained from an E13.5 embryo. Wt1^cre-YFP cells seem to be migrating into the endoderm through discontinuities of the basal lamina revealed by lack of laminin immunoreactivity (arrows in B and D). However, at more posterior levels of the same embryo (C) the basal lamina of the endoderm (arrows) is continuous and no Wt1^cre-YFP cells are present in the mucosa. E,F. Laminin immunoreactivity shows a continuous endodermal basal lamina in areas lacking of Wt1^cre-YFP cells within the mucosa (F, neonate). However, where Wt1^cre-YFP cells are still present in the endoderm, the lamina basal seems to be still discontinuous (arrows in E, E18.5 embryo). Scale bars = 25 µm.</p